Advances in understanding grapevine downy mildew: From pathogen infection to disease management

Abstract Plasmopara viticola is geographically widespread in grapevine‐growing regions. Grapevine downy mildew disease, caused by this biotrophic pathogen, leads to considerable yield losses in viticulture annually. Because of the great significance of grapevine production and wine quality, research on this disease has been widely performed since its emergence in the 19th century. Here, we review and discuss recent understanding of this pathogen from multiple aspects, including its infection cycle, disease symptoms, genome decoding, effector biology, and management and control strategies. We highlight the identification and characterization of effector proteins with their biological roles in host–pathogen interaction, with a focus on sustainable control methods against P. viticola, especially the use of biocontrol agents and environmentally friendly compounds.


| INTRODUC TI ON
Grapevine (Vitis vinifera) has established a deep connection with human culture in its long history spanning over 5000 years (Nascimento et al., 2019).Nowadays, grapevine is one of the most widely distributed fruit crops all around the world and comprises many varieties for wine production, table grapes and raisins for human consumption (Brilli et al., 2018;Xiang et al., 2021).Because of the huge market for these commodities, the grapevine industry is of great importance to economic expansion and increasing income in many countries and areas, with a global market size of over €29 billion (Nascimento et al., 2019).
Grapevines are susceptible to numerous pathogenic microorganisms, leading to various diseases.Downy mildew disease, caused by the obligately biotrophic peronosporomycete Plasmopara viticola, is one of the major threats in vineyards and causes huge losses in yield worldwide, especially in viticulture areas with relatively warm and humid climate conditions (Blasi et al., 2011;Yu et al., 2012).

P. viticola was originally endemic on wild Vitis species of North
America and was introduced into the Bordeaux area in 1871, probably with the acquisition of American grapevine rootstocks used as breeding stock for phylloxera resistance (Gessler et al., 2011;Liu, Weng, et al., 2019).Subsequently, P. viticola was detected in the Bordeaux area in 1878 and rapidly spread across Europe, leading to a severe pandemic throughout the continent in the following decade (Boso & Kassemeyer, 2008;Gessler et al., 2011).
In the field, P. viticola can infect all green tissues of grapevine, including leaves, inflorescences, fruit clusters, and young bunches, reducing the assimilation rate through a reduction in green leaf area and an influence on gas exchange in other green leaf tissues, resulting in significant losses in grapevine productivity and quality (Blasi et al., 2011;Moriondo et al., 2005;Yu et al., 2012).Typically, diseased leaves display yellow or reddish-brown lesions on the upper surface, corresponding to white pathogen growth on the lower surface.Sometimes lesions are oily, somewhat angular and are located between the veins.The leaf lesions become brown and die with age (Musetti et al., 2005).
Because of grapevine's ability to be transformed and micropropagated via somatic embryogenesis, as well as its relatively small genome size relative to other perennials, this species has become a potential model organism for fruit crops in scientific research (Velasco et al., 2007).Additionally, P. viticola is considered a good candidate for the study of host adaptation of biotrophic pathogens (Dussert et al., 2016).In the past decade, hot issues of grapevine downy mildew and its cause, P. viticola, such as environmentally friendly control measures, pathogenesis and disease resistance, have received increasing attention and a great deal of progress has been made.In the current paper, we summarize and discuss the advances in understanding P. viticola from multiple aspects, including its infection cycle, effector biology and control measures.

| INFEC TI ON C YCLE
The life cycle of P. viticola comprises an asexual multiplication phase that occurs during the plant vegetative period and a sexual phase that ensures the survival of the pathogen over winter (Díez-Navajas et al., 2007).The primary sources of inoculum in spring derive from overwintering sexual oospores (Jürges et al., 2009;Vercesi et al., 1999).However, a rapid sequence of asexual propagation by sporangia under optimal conditions, such as high humidity and warm temperatures, causes severe epidemics and renders P. viticola a serious threat to viticulture (Jürges et al., 2009).The extremely efficient cycle of asexual propagation is responsible for the rapid spread of P. viticola worldwide (Islam et al., 2011).During the growing season, the asexually formed, lemon-shaped sporangia release four to eight flagellate zoospores that swarm within the water film on the lower surface of the leaf (Jürges et al., 2009;Unger et al., 2007).On susceptible hosts, the motile zoospores are specially targeted to the stomata, where they shed their flagella and encyst (Jürges et al., 2009;Liu et al., 2015).The phenomenon of zoospores locating to the stomata is mediated by host cues (Islam et al., 2011;Kiefer et al., 2002).
The encysted zoospores generate germ tubes that reach into the substomatal cavity, where they dilate into an infection vesicle (Kiefer et al., 2002;Liu et al., 2015;Yu et al., 2012).From the substomatal vesicle, a primary hypha emerges and develops into a mycelium that spreads inside the leaf tissue, extending mainly into the intercellular spaces of the spongy parenchyma and forming haustoria that penetrate the host cell wall (Jürges et al., 2009;Kiefer et al., 2002).Next, masses of hyaline sporangia are produced from sporangiophores at the lower leaf surface and are released and spread via wind currents or raindrops (Kortekamp, 2006).These sporangia start secondary infections as soon as weather conditions are favourable for their development and if protection is omitted (Kortekamp, 2006).At the end of autumn, numerous oospores, which represent the resting spores of P. viticola, form within fallen leaves and berries, allowing P. viticola to overwinter (Kortekamp, 2006).The life cycle of P. viticola is shown in Figure 1.
The differences may be caused by the large number of repetitive elements in their genomes, which could be the co-evolutionary result of P. viticola and grapevine originating from different geographic regions.With respect to pathogenesis, only a couple of pathogenicity-related genes, such as PvCHS1 and PvCHS2 (Werner et al., 2002), have been characterized, even though thousands of protein-coding genes have been predicted (Dussert et al., 2019;Yin, An, et al., 2017).Therefore, the pathogenic mechanism of major P. viticola genes in susceptible genotypes is still poorly understood, which is partially attributed to the strictly biotrophic lifestyle of P. viticola, which makes this pathogen difficult to study in the laboratory (Chen et al., 2020;Liu, Lan, et al., 2018;Liu, Zhang, et al., 2018;Nascimento et al., 2019).Molecular research on the pathogen has therefore mainly focused on identifying secreted efforts during infection and deciphering the underlying mechanisms of grapevine-P.viticola interactions.

| EFFEC TOR B I OLOGY OF P. VITICOL A
Effectors are a large group of secreted pathogenicity-related factors that can manipulate plant defence responses and modulate host cellular process to promote pathogen colonization in host plants (Lo Presti & Kahmann, 2017).Many effectors have been inferred to enter plant cells based on the physical interaction with host R proteins containing the nucleotide-binding sites and leucine-rich repeats (NB-LRR) or the physiological effects, including activating programmed cell death or suppressing the activities of different cell death inducers when expressed intracellularly (Kale & Tyler, 2011).
Effector proteins are classified into two groups based on their final destinations: cytoplasmic and apoplastic.The cytoplasmic effector proteins locate to different intracellular compartments and play various roles during pathogen host-interactions.The apoplastic effectors are well known to inhibit the activities of host-secreted hydrolases or interrupt the functions of host receptors (Ma et al., 2017;Oh et al., 2009;Tian et al., 2004).

| Cytoplasmic effectors
Cytoplasmic effectors belonging to different types have been identified in P. viticola; the most notable effectors are typically characterized as RxLR and CRN (crinkling and necrosis-inducing or Crinkler) proteins (Jiang & Tyler, 2012;Yin, An, et al., 2017).Functional characterization of the two kinds of proteins has been widely performed in P. viticola.

| RxLR proteins
RxLR proteins are defined by a conserved N-terminal motif similar in sequence, position and function to a host translocation signal RXLX(E/D/Q) present in the malaria parasite Plasmodium falciparum that enables delivery of effector proteins into human erythrocytes (Hiller et al., 2004;Schornack et al., 2010).Usually, the RxLR protein has an N-terminal signal peptide, followed by an RxLR motif or its variant, and an EER motif.
Functional analyses have also uncovered some common or specific characters of RxLR proteins in P. viticola.For example, suppression of plant immunity is the major activity of the RxLR secretome identified in P. viticola (Table 1), a feature shared by the RxLR secretome of H. arabidopsidis (Fabro et al., 2011;Pel et al., 2014).This is reasonable as the biotrophic oomycetes, including P. viticola, may have evolved to generate a relatively high percentage of RxLR effectors to suppress elicitin-triggered cell death, keeping the host tissue alive for sustainable access to nutrients (Xiang et al., 2016).This inference was further exemplified by the inhibitory regulation among RxLR effectors shown in Figure 2.Moreover, the elicitor activity of RxLR proteins is associated with grapevine species, which can be supported by the fact that protein RxLR_PVITv1008311 without a signal peptide from P. viticola isolate PvitFEM01 elicits a cell death response in resistant Vitis riparia but not in susceptible grapevine V. vinifera (Brilli et al., 2018).The different responses may result from the differences in the recognition of P. viticola effectors between the two grapevines.
Although a number of RxLR effectors have been identified, the regulatory mechanisms between RxLR effectors with their interactive targets from plant cells have only been investigated for a couple of effectors.RxLR effectors PvRxLR111 with PvRxLR50253 target and stabilize grapevine proteins VvWRKY40 with VpBPA1 to suppress plant immunity through decreasing H 2 O 2 accumulation and promote pathogen infection (Ma et al., 2021;Yin et al., 2022).
Another RxLR protein PvRxLR131 targets V. vinifera BRI1 kinase inhibitor 1 (VvBKI1) in the plasma membrane as a strategy for promoting infection (Lan et al., 2019).However, it is challenging to determine whether the RxLR proteins have virulence functions in susceptible grape cultivars and characterize their correlations with correspondent resistance (R) proteins, even though the gene-forgene relationship between an avirulent RxLR gene with its cognate resistance (R) gene has been well described in other pathogenic TA B L E 1 The RxLR proteins characterized in Plasmopara viticola.

| CRN proteins
CRN proteins are small, secreted proteins first identified in P. infestans and described as causing a crinkling and necrosis phenotype when ectopically expressed in planta (Torto et al., 2003;Xiang et al., 2021).
CRN proteins have a conserved N-terminal LXLFLAK motif and a conserved HVLVXXP motif followed by variable carboxyl (C)-terminal sequences (Haas et al., 2009;Xiang et al., 2021).
Recent data have revealed that CRN proteins are present in other pathogenic and free-living eukaryotes, including Batrachochytrium dendrobatidis (Sun et al., 2011), Batrachochytrium salamandrivorans (Farrer et al., 2017), Rhizophagus irregularis (Voß et al., 2018), members of Viridiplantae and amoebozoans (Zhang et al., 2016), suggesting that CRN proteins may be more ubiquitously distributed than predicted.However, CRNs are absent in animal-pathogenic oomycetes, suggesting that the evolution and occurrence of this kind of protein may be associated with virulence and adaption on susceptible plants (Gaulin et al., 2018;Voß et al., 2018).
In terms of P. viticola, an array of CRN-like genes have been cloned and characterized from isolates JL-7-2 (Yin, An, et al., 2017), PvitFEM01 (Brilli et al., 2018) and YL (Xiang et al., 2021).Sequence alignments revealed that 27 PvCRN genes from isolate YL share high similarities in nucleotides with their orthologues from another three P. viticola isolates, JL-7-2, INRA-PV221 and PVitFEM01.The high level of intraspecific nucleotide polymorphism among the CRN-like genes from these four P. viticola isolates is considered to be a reflection of pathogen evolution adaptation to different grapevine genotypes in distinct geographic areas (Xiang et al., 2021).Moreover, it was found that gene duplication (PvCRN27 and PvCRN29, PvCRN10 and PvCRN11) and fragment recombination of CRNs occurred during adaptive evolution in P. viticola, which is analogous to the CRN genes in P. sojae (Shen et al., 2013) and P. infestans (Haas et al., 2009).Gene recombination was mainly generated with three different patterns.
In the first pattern, CRN genes contain a highly conserved N-terminal sequence, but differ in the C-terminal sequence; these genes include PvCRN31 and PvCRN11, PvCRN21 and PvCRN22, PvCRN12, PvCRN35 and PvCRN17.The second recombination pattern is that CRN genes have a highly conserved C-terminal sequence but display diversity in the N-terminal coding sequence, such as PvCRN1, PvCRN4 and PvCRN30.Finally, a novel CRN gene is composed of distinct fragments from at least two other PvCRN genes, which can be evidenced The regulatory pathway of RxLR and CRN proteins in Plasmopara viticola.① RxLR proteins interact with or stabilize its target proteins.② Cell death triggered by the proteins depends on SGT1, Hsp90, RAR1 and MAPK cascades.③ Target proteins function as a negative regulator in plant immunity.④ The PvCRN17 competes with VCIA1 to bind with VAE7L1 to suppress Fe-S proteinsmediated defence responses.
by the genes PvCRN15 and PvCRN16 (Xiang et al., 2021).To some extent, this phenomenon may explain the CRN family expansion and sequence divergence of the three oomycetes when compared to other fungi and oomycetes.
Although the first CRN protein was characterized as a crinkling and necrosis-inducing factor on expression in planta, a characteristic of plant innate immunity (Haas et al., 2009;Torto et al., 2003), an array of studies has revealed that this is not a common feature for CRN proteins or their C-terminal domain, and one set of CRN proteins even displays the opposite function.For example, PsCRN63 (Liu, Ye, et al., 2011), PcCRN4 (Mafurah et al., 2015), PiCRN8 (van Damme et al., 2012) and PcCRN83_152 (Amaro et al., 2018) induce cell death, but some other CRN effectors, such as VmEP1 (Li et al., 2015), PsCRN115 (Liu, Ye, et al., 2011), PsCRN70 (Rajput et al., 2014) and PsCRN161 (Rajput et al., 2015), suppress cell death triggered by other elicitins, indicating that CRN proteins possess diverse functions beyond cell death induction (Amaro et al., 2018;Stam et al., 2013;Voß et al., 2018).In P. viticola, most characterized PvCRN proteins suppress or attenuate cell death triggered by other elicitins when transiently expressed in Nicotiana benthamiana (Table 2), which is also inconsistent with the initially documented roles of CRN proteins.Additionally, although many PvCRN genes have been identified in P. viticola, the molecular functions have been explained for only a couple of PvCRN genes.For example, the CRN protein PvCRN17 competed with VCIA1 to bind with VAE7L1, demolishing the cytosolic iron-sulphur (Fe-S) cluster assembly (CIA) Fe-S cluster transfer complex to suppress Fe-S protein-mediated defence responses (Figure 2).In future, the most important and urgent task is to identify the host targets of PvCRN effectors and investigate their molecular functions, which drives the identification of unknown components in plant immunity and metabolism, as well as promoting biotechnology innovations.

| YxSL[RK] proteins
Besides RxLR and CRN motifs, YxSL[RK] is another conserved motif that has been identified in the secreted and non-secreted proteins of oomycete species including P. ultimum (Lévesque et al., 2010), P. infestans and P. sojae (Adhikari et al., 2013), and S. parasitica (Jiang et al., 2013).The YxSL[RK] motif shares similarity in sequence and position with the canonical RxLR motif and appears to be a signature for a novel family of secreted proteins that function as effectors (Adhikari et al., 2013;Lévesque et al., 2010).However, whether the YxSL[RK] motif defines a host-translocation domain as noted for RxLR effectors remains to be determined (Lévesque et al., 2010).

| Apoplastic effectors
Oomycetes not only secrete large numbers of typical RxLR and CRN effectors targeted to the host cytoplasm to alter host physiology and facilitate pathogen colonization, they also release an extensive range of apoplastic effectors that interact with extracellular targets and surface receptors to facilitate infection (Jiang & Tyler, 2012;Yin et al., 2015).The genome of sequenced oomycetes has revealed large complex families of apoplastic effectors, including secreted hydrolytic enzymes such as lyases, proteases, lipases and glycosylases that probably degrade plant tissue, enzyme inhibitors to protect against host defence enzymes, necrotizing toxins such as necrosis-and ethylene-inducing peptide-like proteins (NLPs), Phytophthora cactorum factors and secreted cysteine-rich proteins, that are implicated in pathogenesis during symptom development (Haas et al., 2009;Jiang & Tyler, 2012;Tyler et al., 2006).
The first characterized member of NLP family, the Nep1 protein, was isolated from culture filtrates of Fusarium oxysporum (Bailey, 1995).Experimental tests demonstrated that Nep1 was capable of inducing ethylene biosynthesis as well as necrosis in a wide variety of Dicotyledoneae but not in Monocotyledoneae (Bailey, 1995).Since then, more NLPs have been identified in various phytopathogenic microorganisms, including fungi, bacteria and oomycetes (Xiang et al., 2022).According to the induced phenotypes, NLPs can be classified into two groups: the cytotoxic NLPs, which are able to permeabilize the cellular membrane of dicotyledonous plants and cause necrosis as well as a myriad of other defence responses (Seidl & Van den Ackerveken, 2019), or the noncytotoxic NLPs, with the ability to activate cell death-independent immunity (Seidl & Van den Ackerveken, 2019;Xiang et al., 2022).It was assumed that obligately biotrophic pathogens generally contained the noncytotoxic NLPs, as the biotrophs rely on living plant tissues for their growth and reproduction (Schumacher et al., 2020;Seidl & Van den Ackerveken, 2019).
However, functional analyses of this kind of protein from obligate biotrophs were only performed on a couple of NLPs in P. viticola (Askani et al., 2021;Schumacher et al., 2020;Xiang et al., 2022) and H. arabidopsidis (Cabral et al., 2012).For example, a few NLP genes have been identified in P. viticola and most were highly expressed during the early stages of infection, suggesting that these genes may play major roles during pathogen penetration or initial colonization inside host tissues (Askani et al., 2021;Schumacher et al., 2020;Xiang et al., 2022).However, whether PvNLP genes contribute to virulence for P. viticola is still unknown.Several tested NLPs (PvNLP1-8) are known to be unable to cause necrosis in N. benthamiana (Askani et al., 2021;Schumacher et al., 2020), which is in line with the noncytotoxic effect of tested NLPs of H. arabidopsidis (Cabral et al., 2012).Conversely, Xiang et al. (2022) recently reported that PvNLP7 was able to cause necrosis and en- some of the NLPs suppressed plant growth and enhanced plant resistance against downy mildew, which implies that these NLPs may play roles in different ways independent of necrosis.

| RNA
Apart from effector proteins, bidirectional cross-species small RNA (sRNA)-mediated gene regulation during the compatible interaction has also been revealed in P. viticola.The sRNAs generated by P. viticola trigger the cleavage of grapevine genes and, vice versa, the sRNAs processed from grapevine transcripts target P. viticola messenger RNAs (Brilli et al., 2018).The shuffling of low molecular weight RNA between P. viticola with its host implies that bidirectional communication of sRNAs is an important invasion or resistance strategy adopted by both organisms during the infection.However, the bidirectional exchange pathway and mechanism of sRNAs have not yet been revealed.

| MANAG EMENT AND CONTROL S TR ATEG IE S
In the history of grapevine downy mildew disease management, an array of commodities aimed at killing the pathogen directly or activating induced system resistance indirectly has been developed and widely used in the field.Based on their composition and physicochemical properties, these commodities can be classified into two groups: chemical fungicides and biological control agents.
Alternative measures aimed to reduce the use of chemical fungicides but retain good control over the causal agent, including breeding disease-resistant grapevine varieties and the use of resistance inducers, have received more attention during recent years.Here, we briefly summarize the main developments in these management strategies and discuss their advantages and disadvantages in practical usage.
Because of health and environmental concerns, as well as the detrimental effect on wine quality of long-term use of copper-based fungicides, the usage of cupric fungicides is currently restricted by European Union Regulation 473/2002 and copper-based formulations used in organic farming are limited to 6 kg/ha per year in most European countries (Garde-Cerdán et al., 2017;Kortekamp, 2006).
The development of novel copper-based formulations appears to be a promising approach to enhance control efficiency and minimize the side effects caused by copper (Battiston et al., 2019;La Torre et al., 2010).The advent of nanotechnology provides an innovative perspective to develop pesticides that share slow-release systems to optimize their distribution and persistence, therefore enhancing the protective effect and control efficiency.For example, Cu(II) compounds formulated with synthetic nanostructured hydroxyapatite have resulted in reduced disease severity and higher efficacy even under rain-washed conditions (Battiston et al., 2018(Battiston et al., , 2019)).Although promising results were achieved by the engineered nanoparticles, it is necessary to evaluate the cytotoxicity and genotoxicity of such particles within the plant tissues, as some reports have claimed adverse effects of these nanoparticles on the growth and development of tested plants (Lee et al., 2008;Lin & Xing, 2008).Besides new formulations, the development of alternative strategies to reduce the use of classic chemical fungicides for grapevine downy mildew protection also seems to be an urgent and promising task.
Although many biocontrol microorganisms display a control effect against downy mildew under experimental conditions, the use of these biocontrol agents in agriculture is still far from widespread because of the unmanageable and changeable abiotic stresses (Roatti et al., 2013).Various factors, including environmental factors, production cost, the time period that microorganisms can be stored in packaging, their survival and activity on the plant and in soils, can comprehensively impact the control efficiency (Perazzolli et al., 2012;Roatti et al., 2013;Segarra et al., 2015).A growing amount of research has revealed that appropriate formulation helps to enhance the efficiency of biocontrol agents.For example, application of L. capsici AZ78 in combination with a low dose of a copper-based fungicide leads to higher control efficiency against grapevine downy mildew (Puopolo et al., 2013).Use of L. capsici AZ78 together with corn steep liquor, lignosulfonate and polyethylene glycol in the formulation improves the survival of L. capsici AZ78 cells by one order of magnitude and ensures a high level of protective efficacy (Segarra et al., 2015).T. harzianum T39-induced resistance is attenuated by the combined abiotic stress of heat and drought (Roatti et al., 2013), therefore the optimized formulation is a crucial step in biopesticide development and is an efficient way to maintain persistence in terms of biological control under field conditions.
F I G U R E 3 Different methods used to control Plasmopara viticola.

| Pathogen-resistant grapevine breeding
Grapevine distributed in different geographic areas exhibited susceptibility and resistance against P. viticola at various levels.Generally, the level of grapevine resistance to P. viticola is divided into five classes: immune, extremely resistant, resistant, partly resistant and susceptible (Yu et al., 2012).All the traditional cultivars of V. vinifera, which is the most widely cultivated grapevine species and suitable for wine and table grape production, are susceptible to downy mildew, although variations of susceptibility are observed among different cultivars or even between clones of the same variety (Blanc et al., 2012;Blasi et al., 2011;Boso et al., 2014).In contrast, the North American and Asian Vitis species belonging to the Euvitis subgenus or Muscadinia subgenus exhibit variable levels of resistance to P. viticola, ranging from moderate resistance, such as V. rupestris, to high resistance, including V. rubra, V. candicans, V. amurensis, V. riparia, V. cinerea and Muscadinia rotundifolia (Blasi et al., 2011;Gessler et al., 2011).In nature, control of downy mildew on these traditional grapevine varieties generally relies on the massive use of pesticides (Peressotti et al., 2010).However, routine use of fungicides is becoming increasingly restrictive because of their heavy cost to grapevine production, high risk to human health and adverse impacts on environment (Blanc et al., 2012;Peressotti et al., 2010).Moreover, a growing number of fungicide-resistant P. viticola strains have been detected in the vineyard, reducing the efficiency of fungicide application (Blanc et al., 2012).Therefore, the search for alternative methods to control the disease is important for viticulture (Peressotti et al., 2010).In this context, plant breeding for disease resistance based on the introgression of resistance traits from ancestral species into domesticated varieties appears to be an attractive and environmentally friendly way to control grapevine downy mildew (Blanc et al., 2012;Vezzulli et al., 2019).During the last 20 years, research on the genetic basis of resistance varieties has seen great progress.

| Spray-induced gene silencing of pathogen genes
The crosstalk of sRNA between plant hosts with their fungal and oomycete pathogens has been investigated in some pathosystems (Brilli et al., 2018;Wang et al., 2016), providing new insight into disease management in crops.For example, external application of double-stranded (ds)RNA has been developed as a promising tool to protect plants against various pathogens, such as Fusarium graminearum (Koch et al., 2016), Sclerotinia sclerotiorum (McLoughlin et al., 2018) and Botrytis cinerea (Nerva et al., 2020).In grapevine, the application of dsRNA PvDCL1/2 displayed both protective and curative properties against P. viticola (Haile et al., 2021).These tests provide promising tools by which RNA-based resistant plants or agrochemical alternatives for plant disease management can be developed.However, the mechanism behind the uptake and transport of externally applied dsRNA into host plants remains unclear.

| Natural compounds for disease control
Except for the aforementioned measures adopted to control grapevine downy mildew disease, various types of nature or synthetically produced compounds, including carbohydrate polymers, lipids and (glyco)peptides, that exhibit toxic or prohibitive effects on P. viticola infection have been developed and applied alone or with other copper-based formulations to control downy mildew in grapevine (Garde-Cerdán et al., 2017).Here, we review the inhibitory effects and functional mechanisms of previously characterized compounds.
Although laminarin is able to elicit defence responses in grapevine, protection against P. viticola is unsatisfactory, which could result from the low penetration rate of hydrophilic compounds into the leaf, and laminarin acts solely as an elicitor of plant defence rather than as a toxic compound against oomycetes (Paris et al., 2019).

| PS3
PS3, a sulphated derivative of laminarin, is considered to be the most efficient polysaccharidic resistance inducer against grapevine downy mildew among the reported elicitors (Héloir et al., 2018).PS3 triggers grapevine resistance via a priming phenomenon in which the compound does not elicit classical early signalling events but triggers an enhanced and prolonged plasma membrane depolarization in grapevine cells and causes much more effective resistance against downy mildew (Chalal et al., 2015;Gauthier et al., 2014).The difference in defence responses triggered by laminarin and PS3 may result from the distinct systems evolved by plants to perceive the two compounds (Ménard et al., 2004).Recently, some reports have claimed that the formulation of resistance inducers plays a critical role in their cuticular diffusion and control efficacy against plant diseases.For example, the penetration efficacy of PS3 through leaf cuticle, stomata, anticlinal cell walls and trichomes can be enhanced by a highly ethoxylated surfactant Dehscofix CO125 (DE) and its content is much higher on the abaxial surface of the leaf than on the adaxial surface, which is helpful to guide its practical use in the field (Paris et al., 2016).

| Essential oils
Essential oils (EOs) are another efficient and promising natural protection alternative (Rienth et al., 2019).Terpenes and terpenoids are the main categories of EO compounds and other rare categories including nitrogen-and sulphur-containing compounds, coumarins and homologues of phenylpropanoids (Nazzaro et al., 2017).The antimicrobial activity of EOs might be caused by the properties of terpenes/terpenoids, which are capable of disrupting the cell membrane, causing cell death or inhibiting the sporulation and germination of fungi (Nazzaro et al., 2017;Rienth et al., 2019).A growing amount of evidence indicates that the efficiency of EOs tested in the greenhouse is usually inconsistent with that in the field, which may be attributed to EO degradation caused by light, heat, oxygen, humidity, metal contaminant, application time and poor rain-fastness (Rienth et al., 2019;Turek & Stintzing, 2013).For example, grapevine treated with sage extract (Salvia officinalis) provides a high level of sustained disease control efficacy against P. viticola.However, due to the degradation caused by long-term rainfall, the control efficiency can be significantly reduced in rainy years (Dagostin et al., 2010).inducer to protect a wide range of plant species against biotic and abiotic stresses (Cohen, 2002;Hamiduzzaman et al., 2005;Zimmerli et al., 2008).In penetrated plant cells BABA is thought to block the translocation of nutrients into the haustoria, thereby inhibiting mycelial growth and sporangial production (Hamiduzzaman et al., 2005;Steiner & Schönbeck, 1997).However, BABA-mediated resistance in plants is most probably based on the priming mechanism rather than direct antimicrobial activities (Conrath et al., 2002;Hamiduzzaman et al., 2005;Ton et al., 2005).In response to P. viticola, BABA primes the production of NADPH oxidase-dependent reactive oxygen species and the deposition of callose and lignin (Dubreuil-Maurizi et al., 2010;Hamiduzzaman et al., 2005).However, BABA does not elicit typical defence-related early signalling events such as any variation of cytosolic calcium content, nitric oxide production, reactive oxygen species production, mitogen-activated protein kinase (MAPK) phosphorylation and defence-related gene expression in grapevine cells (Dubreuil-Maurizi et al., 2010).

| Chitosan
Chitosan, a totally or partially deacetylated derivative of chitin, confers high protection against grapevine diseases caused by B. cinerea and P. viticola (Aziz et al., 2006;Romanazzi et al., 2002;Trotel-Aziz et al., 2006).The polycationic β-1,4-linked-d-glucosamine polymer forms a semipermeable film that functions as a physical barrier around infection sites, thereby inhibiting pathogens and inducing defence responses in the host tissues (Garde-Cerdán et al., 2017;Krzyzaniak et al., 2018).It is thought that the activity of chitosan results from its binding to membrane receptors and is dependent on the molecular weight and the degree of N-acetylation (Aziz et al., 2006;Kauss et al., 1989).In grapevine, treatment with chitosan triggers a variety of defence reactions, including the stimulation of lipoxygenase, phenylalanine ammonia-lyase, chitinase and β-1,3-glucanase activities as well as the accumulation of phytoalexins and pathogenesis-related proteins (Aziz et al., 2006;Trotel-Aziz et al., 2006).

| CON CLUS ION
P. viticola has become a serious threat to the viticulture globally.A comprehensive insight into the infection strategies and conditions (such as temperature and humidity), pathogenicity mechanism and plant defence response contributes to establish efficient disease management strategies against downy mildew.Decoding the genome of P. viticola helps researchers to identify and characterize key pathogenicity-related genes that potentially serve as chemical fungicide targets.Revealing the pathogen-host interactive regulation also facilitates the matching of avirulence and resistance genes, which provides important genetic resources for disease resistance breeding.Identification of plant-derived chemical compounds also provides large numbers of valuable and attractive candidates for the development of environmentally friendly fungicides.
Even though great progress in downy mildew control has been made, there is still work to be done to overcome obstacles in plant breeding and disease management.For example, the high risk of P. viticola breaking R gene-mediated resistance and the sustainability of resistant grapevine varieties makes it a challenging project to incorporate multiple resistance genes into susceptible species to extend the resistance duration without the loss of desirable phenotypic traits in grapevine breeding.Moreover, appropriate formulations are urgently required to maintain the duration and efficiency of plant-derived compounds against P. viticola.

ACK N O WLE D G E M ENTS
This study was supported by the Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences grant JKZX202204, the Beijing Talent Program for J.Y. and the National Technology System for Grape Industry CARS-29.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors are not aware of any affiliations, memberships, funding or financial holdings that might be perceived as affecting the objectivity of this review.

F
I G U R E 1 The infection cycle of Plasmopara viticola.Pathogen structures are shown in black.| 3 of 21 PENG et al.
The symbol '-' indicates the localization of these RxLR proteins was undetermined.
hance P. capsici resistance in N. benthamiana with H. arabidopsidis resistance in Arabidopsis.Further research is necessary to resolve these controversial issues.Additionally, even though the major NLPs identified in P. viticola displayed noncytotoxic phenotypes, TA B L E 2 The CRN proteins characterized in Plasmopara viticola.cytoplasm, and nucleus Suppresses BAX-but not INF1-triggered cell death Xiang et al. (2021) PvCRN14 YL Plasma membrane, cytoplasm, and nucleus Suppresses BAX-but not INF1-triggered cell death Xiang et al. (2021) PvCRN15 YL Plasma membrane and the nuclear envelope Suppresses BAX-but not INF1-triggered cell death Xiang et al. cytoplasm, and nucleus Neither suppresses INF1-and BAX-triggered cell death nor induces cell death Xiang et al. (2021) PvCRN35 YL Plasma membrane and the nuclear envelope Suppresses BAX-but not INF1-triggered cell death Xiang et al. (2021) | 11 of 21 PENG et al.